This is a competing renewal application in which we request support for our studies of retrovirus assembly. In the next grant period, we will address two important mechanistic questions relating to the cell biology of retroviral particle assembly. Specific Aim 1: is to understand how retroviral Gag proteins are targeted to specific sites in various cell types and the purpose of the myristoyl-switch in retarding HIV-1 Gag membrane binding. We will first refine and elaborate our biochemical and real-time live cell imaging assays of retrovirus assembly to determine the route that HIV-1 Gag takes to sites of assembly and egress in various cell types, including primary macrophages. These assays, should enable us to determine how HIV-1 Gag proteins select and move to sites of particle assembly. We will test the hypotheses that active transport pathways, or the intrinsic membrane binding properties of Gag, or both, govern Gag targeting. Additionally, we will determine the consequences for HIV-1 assembly and egress of targeting Gag to distinct cellular membranes in various cell types. Moreover we will test several hypotheses as to why HIV-1 has evolved a mechanism that inhibits membrane binding. Specific Aim 2: is to determine how HIV-1 Vpu affects the targeting of retroviral Gag proteins and promiscuously affects enveloped viral particle release. At present, the mechanism by which Vpu facilitates virus release is almost completely unclear. However our preliminary studies reveal dramatic relocalization of HIV-1 and MLV Gag proteins to the plasma membrane in the presence of Vpu. We will undertake a comprehensive mutagenesis approach to map the determinants of Vpu localization, and to determine the importance of Vpu localization and its effects on Gag localization in the stimulation of virus release virus release. We will also determine the underlying mechanism by which Vpu causes relocalisation of retroviral Gag proteins Finally we will attempt to identify cellular targets and/or cofactors of Vpu's virus release function using tandem affinity purification. Understanding how HIV-1 and other retroviral Gag proteins select sites with the cell at which to assemble, and how viral and cellular activities influence this process, remains one of the most enigmatic problems in retrovirology. The experiments proposed herein should provide greater clarity as to how Gag proteins behave during assembly in cells.